Development of a high-throughput flow cytometric neutralization assay to screen for human enterovirus A71 (EVA71) neutralizing antibodies.

The conventional method for screening neutralizing antibodies to human enterovirus A71 (EVA71) (microneutralization assay) is time consuming and requires an expert to perform manual evaluation. An automated neutralization assay could shorten the testing time, improve reproducibility, and provide automatic analysis. This study aimed to develop a high-throughput flow cytometric neutralization assay to screen for EVA71 neutralizing antibodies, and to develop quality control materials to ensure accurate testing. The results of this study demonstrate the high potential viability of the proposed flow cytometric method. Compared to the standard method, the flow cytometric method was shown to require a smaller sample volume, provide a much faster turnaround time, provide a rapid result for interpreting the neutralizing antibody level, and allow for possible quantification of results. The observed drawbacks of the proposed method include higher cost per test, longer hands-on time, and lower sensitivity in low titer conditions, which could lead to false negative results. The developed quality control materials were demonstrated to be effective and storable for 1 month. These results pave the way for the optimization and implementation of an automated neutralization assay to screen for neutralizing antibodies not only against EVA71, but also against other viruses in the enterovirus genus.